A functional skeletal system depends upon the coordinated development of cartilages and joints during embryogenesis. However, little is known about the cellular and molecular mechanisms that control cartilage size, shape or connectivity. Progress unraveling the signals that direct mesenchymal cells to condense and align into prechondrogenic stacks is of particular importance in elucidation of the early events that shape the organization and growth of the skeleton. Understanding these processes will allow better diagnostic approaches and treatments for skeletal malformations and birth defects. Moreover, molecules that control cartilage morphogenesis and differentiation may be of considerable clinical importance both for improvements in the treatment of cartilage and joint injuries and in bioengineering efforts to induce cartilage formation from stem cells. Our recent finding that Rere, a close relative of the transcription factor Atrophin which regulates Dachsous (Dchs)-Fat signaling in flies, is required for craniofacial development in zebrafish has implicated the Fat pathway in planar cell polarity (PCP) during skeletogenesis. Dramatic results from our laboratory now demonstrate an even more profound role for a Fat3 in cartilage differentiation, consistent with PCP. Embryos deficient in Rere or Fat3 develop cartilages in which stacking and differentiation are uncoupled in the pharyngeal arches and this leads to joint fusion. Moreover, Rere and Fat3 are critical for coordinating responses of skeletal progenitors to Bmp and Fgf signaling during craniofacial development. We propose to explore the roles of the Fat pathway and PCP in cartilage and joint formation in three sets of experiments. The first aim will address the hypothesis that the Fat pathway couples cartilage stacking and differentiation. Cartilage and joint phenotypes will be evaluated in Rere and Fat3 mutants using live imaging. We will identify the Dchs ligand(s) for Fat in cartilage and create ectopic sources to study signal propagation and modulation. We will also compare the activities of the Fat pathway and Wnt/PCP and their interdependence in cartilage development. The second aim will address the hypothesis that the Fat pathway coordinates polarized responses to Bmps and other growth factors, thereby linking early patterning events with later organ formation. For this we have new markers of cytoskeletal polarity and cilia that reveal unexpected boundaries of polarity in skeletal progenitors. Finally, the third aim will focus on joint formation and test the hypothesis that the Fat pathway prevents cartilage stacking and differentiation in the joint interzone with inducible elimination or overexpression of Rere or Fat3. Together these studies will lead to mechanistic insights into the relatively unexplored functions of the vertebrate Fat pathway in cell-cell communication and identify candidate genes for as yet unresolved causes of human congenital skeletal malformations. PUBLIC HEALTH RELEVANCE: The proposed studies will provide some of the first evidence that the Fat pathway influences skeletogenesis. Defining molecules that control cartilage development may be of clinical importance both for improving treatments for cartilage and joint injuries and in efforts to induce cartilage from stem cells. The Fat pathway component Rere is also implicated in dentatorubral-pallido-luysian atrophy and understanding its functions could help improve diagnosis and treatments for this and related neurodegenerative diseases.